Developmental expression of the rat muscle glycogen phosphorylase gene is not directly regulated by the four muscle regulatory factor proteins

The muscle isozyme of glycogen phosphorylase (MGP)is developmentally and neurally regulated in rat muscle. A series ot deletion derivatives were generated in a CAT reporter vector to identify elements in the 5' noncoding region of MGP required for developmental regulation. A 269 bp fragment was identified that conferred 15-fold higher levels of CAT activity in C2C12 myotubes than myoblasts. This fragment contained four E box motifs located at -167, -118, -18 and +29, a CArG-like element (CCAAAATAGC) at -109, a GC rich region at -63 and a TATA motif 27 bp 5' of the transcriptional start point. Point mutations of the E boxes had little affect on the developmental regulation of MGP. Three of the muscle regulatory factor proteins (MyoD, myogenin and MRF4) transactivated the MGP promoter by only 2-fold and one them ( myf-5) had no effect on CAT activities in cotransfection studies in the nonmuscle cell line C3H10T1/2. Deletion of the CArG-like sequence reduced the difference between MT and MB CAT activity to only 1.8-fold. This may be a potential area of importance in the developmental-specific regulation of MGP in muscle cells. Further deletion of the GC motif eliminated any measurable CAT activity and defined the minimal promoter region between -76 and +3.